The bifunctional enzyme, 6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase, is a crucial element in the regulation of hepatic glucose levels. It is the sole enzyme responsible for the synthesis and degradation of fructose2,6-bisphosphate, a potent, intracellular modulator of carbohydrate metabolic flux. Fructose-2,6-bisphosphate is an allosteric activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase. The metabolic effects of glucagon on hepatic carbohydrate fluxes are mediated by changes in the activity of this bifunctional enzyme. The central role of fructose 2,6-bisphosphate in the control of hepatic glucose metabolism suggests that drug therapies directed toward increasing the fructose-2,6-bisphosphate levels in the liver may be beneficial to the diabetic patient. In this proposal, we plan to obtain high resolution x-ray crystallographic structural information about the substrate and regulatory sites of this enzyme system with the hope that these studies will provide a basis for the development of useful pharmacological agents. Two basic experimental approaches are described: I) Determine the structure of complexes of the bisphosphatase domain with various substrates and inhibitors, e.g. GTP, guanosine, Fructose-6-P, substrate analogs, and a 30 amino acid peptide inhibitor. II) The liver bifunctional enzyme is physiologically modulated by phosphorylation near the amino terminal end of the kinase domain. We will investigate the structure-function relations that are the basis of this modulation by determining the structure of the human liver bifunctional enzyme in the phosphorylated and dephosphorylated state.